pcDNA 6/myc-His A, B, and C - Thermo Fisher Scientific · PureLink™ HiPure Plasmid Miniprep Kit 100 preps K2100-03 PureLink ... testing. The sequence of pcDNA™6/myc-His A is available

User Manual

Corporate HeadquartersInvitrogen Corporation1600 Faraday AvenueCarlsbad, CA 92008T: 1 760 603 7200F: 1 760 602 6500E: [email protected]

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pcDNA™6/myc-His A, B, and C

Catalog no. V221-20

Rev. date: 9 November 2010

Manual part no. 25-0233

MAN0000076

ii

iii

Table of Contents

Kit Contents and Storage..................................................................................................................................... iv

Accessory Products ............................................................................................................................................... v

Introduction ................................................................................................................... 1

Overview .................................................................................................................................................................1

Methods ......................................................................................................................... 2

Cloning into pcDNA™6/myc-His A, B, and C.....................................................................................................2

Transfection and Analysis.....................................................................................................................................7

Creating Stable Cell Lines ...................................................................................................................................10

Appendix...................................................................................................................... 14

pcDNA™6/myc-His Vector..................................................................................................................................14

pcDNA™6/myc-His/lacZ.....................................................................................................................................16

Technical Support.................................................................................................................................................17

Purchaser Notification .........................................................................................................................................18

References..............................................................................................................................................................19

iv

Kit Contents and Storage

Shipping and Storage

pcDNA™6/myc-His vectors are shipped on wet ice. Upon receipt, store vectors at –20°C.

Kit Contents All vectors are supplied as detailed below. Store the vectors at –20°C.

Vector Composition Amount

pcDNA™6/myc-His A, B, and C pcDNA™6/myc-His/lacZ

40 μl of 0.5 μg/μl vector in 10 mM Tris-HCl, 1 mM EDTA, pH 8.0 40 μl of 0.5 μg/μl vector in 10 mM Tris-HCl, 1 mM EDTA, pH 8.0

20 μg

20 μg

v

Accessory Products

Introduction The products listed below are designed to help you detect and purify your

recombinant fusion proteins expressed from pcDNA™6/myc-His vectors. In addition, Invitrogen has a wide variety of mammalian expression vectors, many of which can be utilized with pcDNA™6/myc-His to express multiple proteins in the same cell (see next page). For more information, www.invitrogen.com or contact Technical Support (see page 17).

Product Amount Catalog no.

ProBond™ Purification System 6 purifications K850-01

50 ml R801-01 ProBond™ Resin

150 ml R801-15

PureLink™ HiPure Plasmid Miniprep Kit 100 preps K2100-03

PureLink™ HiPure Plasmid Midiprep Kit 25 preps K2100-04

Electrocomp™ TOP10F’ 2 × 20 rxns 6 × 20 rxns

C665-11 C665-24

One Shot™ TOP10F’ (chemically competent cells) 20 × 50 μl C3030-03

-Gal Assay Kit 1 kit K1455-01

-Gal Staining Kit 1 kit K1465-01

Blasticidin 50 mg R210-01

Antibodies for Detection

If you do not have an antibody to your protein, Invitrogen offers the Anti-myc antibodies or the Anti-His(C-term) antibodies to detect your recombinant protein. Horseradish peroxidase (HRP)-conjugated antibodies are available for convenient one-step detection.

Antibody Epitope Catalog no.

Anti-myc R950-25

Anti-myc-HRP

Detects 10 amino acid epitope derived from c-myc (Evans et al., 1985): EQKLISEEDL

R951-25

Anti-His(C-term) R930-25

Anti-His(C-term)-HRP

Detects the C-terminal polyhistidine tag (requires the free carboxyl group for detection) (Lindner et al., 1997): HHHHHH-COOH R931-25

Other Mammalian Expression Vectors

We have a wide variety of mammalian expression vectors utilizing the CMV or EF-1 promoters. Vectors are available with the Xpress™ (N-terminal), c-myc (C-terminal), V5 (C-terminal), or C-terminal polyhistidine epitopes for detection and either the neomycin, blasticidin, or Zeocin™ resistance genes. All vectors utilize the polyhistidine tag for purification using ProBond™ resin. For more information on the mammalian expression vectors available, see our website (www.invitrogen.com) or call Technical Support (page 17).

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1

Introduction

Overview

Description of the System

pcDNA™6/myc-His A, B, and C are 5.1-kb vectors designed for overproduction of recombinant proteins in mammalian cell lines. Features of the vectors allow purification and detection of expressed proteins (see pages 14–15 for more information). High-level stable and transient expression can be carried out in most mammalian cells. The vectors contain the following elements:

Human cytomegalovirus immediate-early (CMV) promoter for high-level expression in a wide range of mammalian cells.

Three reading frames to facilitate in-frame cloning with a C-terminal peptide encoding the myc (c-myc) epitope and a polyhistidine (6xHis) metal-binding tag.

Blasticidin resistance gene (bsd) for selection of stable cell lines (Kimura et al., 1994).

Episomal replication in cell lines that are latently infected with SV40 or that express the SV40 large T antigen (e.g., COS7).

The control plasmid, pcDNA™6/myc-His/lacZ, is included for use as a positive control for transfection, expression, and detection in the cell line of choice.

Experimental Outline

Use the following outline to clone and express your gene of interest in pcDNA™6/myc-His.

Consult the multiple cloning sites described on pages 3–5 to determine which vector (A, B, or C) should be used to clone your gene in-frame with the C-terminal myc epitope and the polyhistidine tag. Ligate your insert into the appropriate vector and transform into E. coli. Select transformants on 50–100 μg/ml ampicillin or 50 μg/ml blasticidin.

Analyze your transformants for the presence of the insert by restriction digestion.

Select a transformant with the correct restriction pattern and use sequencing to confirm that your gene is cloned in-frame with the C-terminal peptide.

Transfect your construct into the cell line of choice using your own method of transfection.

Test for expression of your recombinant gene by western blot analysis or functional assay. For antibodies to the myc epitope or the C-terminal polyhistidine tag, see the next page.

To purify your recombinant protein, you may use metal-chelating resin such as ProBond™. ProBond™ resin is available separately (see page v).

2

Methods

Cloning into pcDNA™6/myc-His A, B, and C

General Molecular Biology Techniques

For help with DNA ligations, E. coli transformations, restriction enzyme analysis, purification of single-stranded DNA, DNA sequencing, and DNA biochemistry, refer to published references (Ausubel et al., 1994; Sambrook et al., 1989).

E. coli Strain Many E. coli strains are suitable for the propagation of pcDNA™6/myc-His

vectors, including TOP10F´, DH5-F’™, JM109 and INVF’. We recommend that you propagate vectors containing inserts in E. coli strains that are recombinant deficient (recA) and endonuclease A-deficient (endA).

For your convenience, TOP10F’ is available as chemically competent or electrocompetent cells from Invitrogen (see page v).

Transformation Method

You may use any method of your choice for transformation. Chemical transformation is the most convenient for most researchers. Electroporation is the most efficient and the method of choice for large plasmids.

Maintenance of pcDNA™6/myc-His

To propagate and maintain the pcDNA™6/myc-His vectors, use the supplied 0.5 μg/μl stock solution in TE, pH 8.0 to transform a recA, endA E. coli strain like TOP10F’, DH5™, JM109 or equivalent.

Select transformants on LB plates containing 50–100 μg/ml ampicillin or 50 μg/ml blasticidin. Be sure to prepare a glycerol stock of each plasmid for long-term storage (see page 6 for recipe).

Cloning Considerations

Your insert should contain a Kozak consensus sequence with an ATG initiation codon for proper initiation of translation (Kozak, 1987; Kozak 1990). An example of a Kozak consensus sequence is provided below. Other sequences are possible, but the G or A at position –3 and the G at position +4 (shown in bold) illustrates the most commonly occurring sequence with strong consensus. Replacing one of the two bases at these positions provides moderate consensus, while having neither results in weak consensus. The ATG initiation codon is shown underlined.

(G/A)NNATGG

If you wish to express your protein WITHOUT the C-terminal peptide, be sure to include a stop codon.

Continued on next page

3

Cloning into pcDNA™6/myc-His A, B, and C, Continued

Multiple Cloning Site of Version A

Below is the multiple cloning site for pcDNA™6/myc -His A. Restriction sites are labeled to indicate the cleavage site. The boxed nucleotides indicate the variable region. Note that there is a stop codon between the BamH I site and the BstX I site. The multiple cloning site has been confirmed by sequencing and functional testing. The sequence of pcDNA™6/myc -His A is available for downloading from our website at www.invitrogen.com or from Technical Support (page 17).

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4


Multiple Cloning Site of Version B

Below is the multiple cloning site for pcDNA™6/myc -His B. Restriction sites are labeled to indicate the cleavage site. The boxed nucleotides indicate the variable region. The multiple cloning site has been confirmed by sequencing and functional testing. The sequence of pcDNA™6/myc -His B is available for downloading from our website at www.invitrogen.com or from Technical Support (page 17).

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Multiple Cloning Site of Version C

Below is the multiple cloning site for pcDNA™6/myc -His C. Restriction sites are labeled to indicate the cleavage site. The boxed nucleotides indicate the variable region. The multiple cloning site has been confirmed by sequencing and functional testing. The sequence of pcDNA™6/myc -His C is available for downloading from our website at www.invitrogen.com or from Technical Support (page 17).

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E. coli Transformation

Transform your ligation mixtures into a competent recA, endA E. coli strain (e.g. TOP10F´, DH5™) and select on LB plates containing 50–100 μg/ml ampicillin or 50 μg/ml blasticidin. Select 10–20 clones and analyze for the presence and orientation of your insert.

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We recommend that you sequence your construct with the T7 Forward and BGH Reverse primers to confirm that your gene is fused in frame with the myc epitope and the C-terminal polyhistidine tag.

Preparing a Glycerol Stock

Once you have identified the correct clone, be sure to purify the colony and make a glycerol stock for long-term storage. It is also a good idea to keep a DNA stock of your plasmid at –20°C.

• Streak the original colony out on an LB plate containing 50 μg/ml ampicillin or 50 μg/ml blasticidin. Incubate the plate at 37°C overnight.

• Isolate a single colony and inoculate into 1–2 ml of LB containing 50 μg/ml ampicillin.

• Grow the culture to mid-log phase (OD600 = 0.5–0.7).

• Mix 0.85 ml of culture with 0.15 ml of sterile glycerol and transfer to a cryovial.

Store at –80°C.

7

Transfection and Analysis

Introduction Once you have confirmed that your construct is in the correct orientation and

fused in frame with the C-terminal peptide, you are ready to transfect your cell line of choice. We recommend that you include the positive control vector and a mock transfection to evaluate your results.

Plasmid Preparation

Plasmid DNA for transfection into eukaryotic cells must be very clean and free from phenol and sodium chloride. Contaminants will kill the cells and salt will interfere with lipid complexing, decreasing transfection efficiency. We recommend isolating plasmid DNA using the PureLink™ HiPure Miniprep Kit or the PureLink™ HiPure Midiprep Kit (see page v).

Methods of Transfection

For established cell lines (e.g., HeLa), consult original references or the supplier of your cell line for the optimal method of transfection. It is recommended that you follow exactly the protocol for your cell line. Pay particular attention to medium requirements, when to pass the cells, and at what dilution to split the cells. Further information is provided in Current Protocols in Molecular Biology.

Methods of transfection include calcium phosphate (Chen & Okayama, 1987; Wigler et al., 1977), lipid-mediated (Felgner et al., 1989; Felgner & Ringold, 1989) and electroporation (Chu et al., 1987; Shigekawa & Dower, 1988). For high efficiency transfection in a broad range of mammalian cells, we recommend using Lipofectamine™ 2000 Reagent available from Invitrogen. For more information on Lipofectamine™ 2000 and other transfection reagents available, visit our website at www.invitrogen.com or contact Technical Support (page 17).

Positive Control pcDNA™6/myc-His/lacZ is provided as a positive control vector for mammalian

cell transfection and expression (see page 16) and may be used to optimize transfection conditions for your cell line. The gene encoding -galactosidase is expressed in mammalian cells under the control of the CMV promoter. A successful transfection will result in -galactosidase expression that can be easily assayed (see below).

Assay for -galactosidase Activity

You may assay for -galactosidase expression by activity assay using cell-free lysates (Miller, 1972) or by staining the cells for activity. Invitrogen offers the -Gal Assay Kit and the -Gal Staining Kit for fast and easy detection of -galactosidase expression (see page v).



8

Transfection and Analysis, Continued

Detection of Fusion Proteins

Several antibodies are available from Invitrogen to detect expression of your fusion protein from pcDNA™6/myc-His (see page v).

To detect fusion protein by Western blot, you will need to prepare a cell lysate from transfected cells. We recommend that you perform a time course to optimize expression of the fusion protein (e.g., 24, 48, 72 hours, etc. after transfection). To lyse cells:

1. Wash cell monolayers (~106 cells) once with phosphate-buffered saline (PBS).

2. Scrape cells into 1 ml PBS and pellet the cells at 1,500 × g for 5 minutes.

3. Resuspend in 50 μl Cell Lysis Buffer (see recipe below). Other lysis buffers may be suitable.

4. Incubate cell suspension at 37°C for 10 minutes to lyse the cells.

5. Centrifuge the cell lysate at 10,000 × g for 10 minutes to pellet nuclei and transfer the supernatant to a fresh tube. Assay the lysate for protein concentration. Note: Do not use protein assays utilizing Coomassie® Blue or other dyes. NP-40 interferes with the binding of the dye with the protein.

6. Add SDS-PAGE sample buffer to a final concentration of 1X and boil the sample for 5 minutes.

7. Load 20 μg of lysate onto an SDS-PAGE gel and electrophorese. Use the appropriate percentage of acrylamide to resolve your fusion protein.

Cell Lysis Buffer 50 mM Tris, pH 7.8

150 mM NaCl 1% Nonidet P-40

1. This solution can be prepared from the following common stock solutions. For 100 ml, combine:

1 M Tris base 5 ml 5 M NaCl 3 ml Nonidet P-40 1 ml

2. Bring the volume up to 90 ml with deionized water and adjust the pH to 7.8 with HCl.

3. Bring the volume up to 100 ml. Store at room temperature.

Note: Protease inhibitors may be added at the following concentrations:

1 mM PMSF 1 μg/ml pepstatin 1 μg/ml leupeptin


9

Transfection and Analysis, Continued

The C-terminal peptide containing the myc epitope and the polyhistidine tag will add approximately 3 kDa to the size of your protein.

Purification You will need 5 × 106 to 1 × 107 transfected cells for purification of your protein

on a 2-ml ProBond™ column (or other metal-chelating column). Refer to the manufacturer's instructions before attempting to purify your fusion protein. To prepare cells for lysis, refer to the protocol on page 13.

10

Creating Stable Cell Lines

Introduction The pcDNA™6/myc-His vectors contain the blasticidin resistance gene for

selection of stable cell lines using blasticidin. We recommend that you test the sensitivity of your mammalian host cell to blasticidin, as natural resistance varies among cell lines. General information and guidelines are provided below for your convenience.

Blasticidin Blasticidin S HCl is a nucleoside antibiotic isolated from Streptomyces

griseochromogenes that inhibits protein synthesis in both prokaryotic and eukaryotic cells (Takeuchi et al., 1958; Yamaguchi et al., 1965). Resistance is conferred by expression of either one of two blasticidin S deaminase genes: bsd from Aspergillus terreus (Kimura et al., 1994) or bsr from Bacillus cereus (Izumi et al., 1991). These deaminases convert blasticidin S to a non-toxic deaminohydroxy derivative (Izumi et al., 1991).

Molecular Weight, Formula and Structure

The formula for blasticidin is C17H26N8O5-HCl and the molecular weight is 458.9. The diagram below shows the structure of blasticidin.

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Handling Blasticidin

Always wear gloves, mask, goggles, and protective clothing (e.g., a laboratory coat) when handling blasticidin. Weigh out blasticidin and prepare solutions in a hood.


11

Creating Stable Cell Lines, Continued

Preparing and Storing Stock Solutions

Blasticidin may be obtained from Invitrogen in 50-mg aliquots (see page v). Blasticidin is soluble in water. Water is generally used to prepare stock solutions of 5–10 mg/ml.

• Dissolve blasticidin in sterile water and filter-sterilize the solution.

• Aliquot in small volumes suitable for one-time use (see last point below) and freeze at –20°C for long-term storage or store at +4°C for short term storage.

• Aqueous stock solutions are stable for 1–2 weeks at +4°C and 6–8 weeks at –20°C.

• The pH of the aqueous solution should not exceed 7 to prevent inactivation of blasticidin.

• Do not subject stock solutions to freeze/thaw cycles (do not store in a frost-free freezer).

Upon thawing, use what you need and discard the unused portion.

Possible Sites for Linearization

To obtain stable transfectants, you may choose to linearize your vector before transfection. While linearizing your vector may not improve the efficiency of transfection, it increases the chances that the vector does not integrate in a way that disrupts the gene of interest. The table below lists unique sites that may be used to linearize your construct prior to transformation. Other restriction sites are possible. Note that the cleavage site is indicated for versions A, B, and C of pcDNA™6/myc-His. Be sure that your insert does not contain the restriction enzyme site you wish to use to linearize your vector.

Enzyme Restriction Site (bp)

(A,B,C) Location Supplier

Bgl II 13 Upstream of CMV promoter Many

Mfe I 162 Upstream of CMV promoter New England Biolabs

Mlu I 229 5´ end of CMV promoter Many

Bst1107 I 2932 (A), 2936 (B), 2928 (C) End of SV40 poly A AGS*, Fermentas, Takara, Boehringer-Mannhiem

Eam1105 I 4204 (A), 4208 (B), 4200 (C) Ampicillin gene AGS*, Fermentas, Takara

Fsp I 4426 (A), 4430 (B), 4422 (C) Ampicillin gene Many

Sca I 4684 (A), 4688 (B), 4680 (C) Ampicillin gene Many

Ssp I 5008 (A), 5012 (B), 5004 (C) Ampicillin gene Many * Angewandte Gentechnologie Systeme


12


Selection in Mammalian Cell Lines

To generate a stable cell line expressing your protein, you need to determine the minimum concentration of blasticidin required to kill your untransfected host cell line. Typically, concentrations between 2 and 10 μg/ml blasticidin are sufficient to kill the untransfected host cell line. Test a range of concentrations (see below) to ensure that you determine the minimum concentration necessary for your cell line.

• Seed cells (2 × 105 cells/60 mm plate) for each time point and allow the cells to adhere overnight.

• The next day, substitute culture medium with medium containing varying concentrations of blasticidin (e.g., 0, 1, 3, 5, 7.5, and 10 μg/ml).

• Replenish the selective medium every 3–4 days. Cells sensitive to blasticidin will round up and detach from the plate. Dead cells will accumulate in the medium.

Count the number of viable cells at regular intervals to determine the appropriate concentration of blasticidin that prevents growth.

Selection of Stable Integrants

Once the appropriate blasticidin concentration is determined, you can generate a stable cell line with your construct. Colonies can generally be identified in 7–10 days with complete selection and expansion in 2 weeks.

Transfect your cells using the appropriate protocol for your cell line. Include a sample of untransfected cells as a negative control.

After transfection, wash the cells once with 1X PBS and add fresh medium to the cells.

48 hours after transfection, split the cells into fresh medium containing blasticidin at the appropriate concentration for your cell line. Split the cells such that they are no more than 25% confluent.

Replenish selective medium every 3–4 days until blasticidin-resistant colonies are detected.

Pick and expand colonies.


13


Preparing Cells for Lysis

Use the procedure below to prepare cells for lysis prior to purification of your protein on ProBond™. You will need 5 × 106 to 1 × 107 cells for purification of your protein on a 2-ml ProBond™ column (see ProBond™ Purification manual).

1. Seed cells in either five T-75 flasks or two-to-three T-175 flasks. 2. Grow the cells in selective medium until they are 80–90% confluent. 3. Harvest the cells by treating with trypsin-EDTA for 2–5 minutes or by

scraping the cells in PBS. 4. Inactivate the trypsin by diluting with fresh medium (if necessary) and transfer

the cells to a sterile microcentrifuge tube. 5. Centrifuge the cells at 1,500 rpm for 5 minutes. Resuspend the cell pellet in

PBS. 6. Centrifuge the cells at 1,500 rpm for 5 minutes. You may lyse the cells

immediately or freeze in liquid nitrogen and store at –80°C until needed.

Lysis of Cells If you are using ProBond™ resin, refer to the ProBond™ Purification manual for

details about sample preparation for chromatography.

If you are using other metal-chelating resin, refer to the manufacturer's instruction for recommendations on sample preparation.

14

Appendix

pcDNA™6/myc-His Vector

Map of pcDNA™6/myc-His

The figure below summarizes the features of the pcDNA™6/myc-His vectors. The sequences for pcDNA™6/myc-His A, B, and C are available for downloading from our website (www.invitrogen.com ) or from Technical Support (see page 17).

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pcDNA™6/myc-His Vector, Continued

Features of pcDNA™6/myc-His

pcDNA™6/myc-His A (5,126 bp), pcDNA™6/myc-His B (5,130 bp), and pcDNA™6/myc-His C (5,122 bp) contain the following elements. All features have been functionally tested.

Feature Benefit

Human cytomegalovirus (CMV) immediate-early promoter/enhancer

Permits efficient, high-level expression of your recombinant protein (Andersson et al., 1989; Boshart et al., 1985; Nelson et al., 1987).

T7 promoter/priming site Allows for in vitro transcription in the sense orientation and sequencing through the insert.

Multiple cloning site in three reading frames

Allows insertion of your gene and facilitates cloning in frame with the myc epitope and polyhistidine C-terminal tag.

myc epitope (Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu)

Allows detection of your recombinant protein with the Anti-myc Antibody or Anti-myc-HRP Antibody (Evans et al., 1985).

C-terminal polyhistidine tag Permits purification of your recombinant protein on metal-chelating resin such as ProBond™. In addition, the C-terminal polyhistidine tag is the epitope for the Anti-His(C-term) Antibody (Lindner et al., 1997) and the Anti-His (C-term)-HRP Antibody.

BGH reverse priming site Permits sequencing through the insert.

Bovine growth hormone (BGH) polyadenylation signal

Efficient transcription termination and polyadenylation of mRNA (Goodwin and Rottman, 1992).

f1 origin Allows rescue of single-stranded DNA.

SV40 early promoter and origin Allows efficient, high-level expression of the blasticidin resistance gene and episomal replication in cells expressing the SV40 large T antigen.

EM-7 promoter Synthetic promoter based on the bacteriophage T7 promoter for expression of the blasticidin resistance gene in E. coli.

Blasticidin resistance gene (bsd) Selection of stable transfectants in mammalian cells (Kimura et al., 1994).

SV40 polyadenylation signal Efficient transcription termination and polyadenylation of mRNA.

pUC origin High-copy number replication and growth in E. coli.

Ampicillin resistance gene (-lactamase)

Selection of transformants in E. coli.

16

pcDNA™6/myc-His/lacZ

Map of pcDNA™6/myc-His/lacZ

pcDNA™6/myc-His/lacZ is a 8173-bp control vector containing the gene for -galactosidase. This vector was constructed by ligating a 3,932-bp BamH I-Stu I fragment containing the blasticidin resistance gene from pcDNA™6/myc-His B to a 4,240-bp BamH I-Stu I fragment containing the lacZ gene, myc epitope, and polyhistidine tag from pcDNA™3.1/myc-His/lacZ.

The figure below summarizes the features of the pcDNA™6/myc-His/lacZ vector.

The nucleotide sequence for pcDNA™6/myc-His/lacZ is available for downloading from our website (www.invitrogen.com) or by contacting Technical Support (see page 17).

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17

Technical Support

Web Resources

Visit the Invitrogen website at www.invitrogen.com for: Technical resources, including manuals, vector maps and sequences, application

notes, MSDSs, FAQs, formulations, citations, handbooks, etc.

Complete technical support contact information

Access to the Invitrogen Online Catalog

Additional product information and special offers

Contact Us For more information or technical assistance, call, write, fax, or email. Additional international offices are listed on our website (www.invitrogen.com).

Corporate Headquarters: 5791 Van Allen Way Carlsbad, CA 92008 USA Tel: 1 760 603 7200 Tel (Toll Free): 1 800 955 6288 Fax: 1 760 602 6500 E-mail: [email protected]

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MSDS Material Safety Data Sheets (MSDSs) are available on our website at www.invitrogen.com/msds.

Certificate of Analysis

The Certificate of Analysis provides detailed quality control and product qualification information for each product. Certificates of Analysis are available on our website. Go to www.invitrogen.com/support and search for the Certificate of Analysis by product lot number, which is printed on the box.

Limited Warranty Invitrogen (a part of Life Technologies Corporation) is committed to providing our customers with high-quality goods and services. Our goal is to ensure that every customer is 100% satisfied with our products and our service. If you should have any questions or concerns about an Invitrogen product or service, contact our Technical Support Representatives. All Invitrogen products are warranted to perform according to specifications stated on the certificate of analysis. The Company will replace, free of charge, any product that does not meet those specifications. This warranty limits the Company’s liability to only the price of the product. No warranty is granted for products beyond their listed expiration date. No warranty is applicable unless all product components are stored in accordance with instructions. The Company reserves the right to select the method(s) used to analyze a product unless the Company agrees to a specified method in writing prior to acceptance of the order. Invitrogen makes every effort to ensure the accuracy of its publications, but realizes that the occasional typographical or other error is inevitable. Therefore the Company makes no warranty of any kind regarding the contents of any publications or documentation. If you discover an error in any of our publications, please report it to our Technical Support Representatives. Life Technologies Corporation shall have no responsibility or liability for any special, incidental, indirect or consequential loss or damage whatsoever. The above limited warranty is sole and exclusive. No other warranty is made, whether expressed or implied, including any warranty of merchantability or fitness for a particular purpose.



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Purchaser Notification

Limited Use Label License No. 22: Vectors and Clones Encoding Histidine Hexamer

This product is licensed under U.S. Patent Nos. 5,284,933 and 5,310,663 and foreignequivalents from Hoffmann-LaRoche, Inc., Nutley, NJ and/or Hoffmann-LaRocheLtd., Basel, Switzerland and is provided only for use in research. Information aboutlicenses for commercial use is available from QIAGEN GmbH, Max-Volmer-Str. 4,D-40724 Hilden, Germany.

Limited Use Label License No. 51: Blasticidin and the Blasticidin Selection Marker

Blasticidin and the blasticidin resistance gene (bsd) are the subject of U.S. PatentNo. 5,527,701 sold under patent license for research purposes only. For inform-ation on purchasing a license to this product for purposes other than research,contact Licensing Department, Life Technologies Corporation, 5791 Van AllenWay, Carlsbad, California 92008. Phone (760) 603-7200. Fax (760) 602-6500.


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References

Andersson, S., Davis, D. L., Dahlbäck, H., Jörnvall, H., and Russell, D. W. (1989). Cloning, Structure, and

Expression of the Mitochondrial Cytochrome P-450 Sterol 26-Hydroxylase, a Bile Acid Biosynthetic Enzyme. J. Biol. Chem. 264, 8222-8229.

Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1994). Current Protocols in Molecular Biology (New York: Greene Publishing Associates and Wiley-Interscience).

Boshart, M., Weber, F., Jahn, G., Dorsch-Häsler, K., Fleckenstein, B., and Schaffner, W. (1985). A Very Strong Enhancer is Located Upstream of an Immediate Early Gene of Human Cytomegalovirus. Cell 41, 521-530.

Chen, C., and Okayama, H. (1987). High-Efficiency Transformation of Mammalian Cells by Plasmid DNA. Molec. Cell. Biol. 7, 2745-2752.

Chu, G., Hayakawa, H., and Berg, P. (1987). Electroporation for the Efficient Transfection of Mammalian Cells with DNA. Nucleic Acids Res. 15, 1311-1326.

Evans, G. I., Lewis, G. K., Ramsay, G., and Bishop, V. M. (1985). Isolation of Monoclonal Antibodies Specific for c-myc Proto-oncogene Product. Mol. Cell. Biol. 5, 3610-3616.

Felgner, P. L., Holm, M., and Chan, H. (1989). Cationic Liposome Mediated Transfection. Proc. West. Pharmacol. Soc. 32, 115-121.

Felgner, P. L., and Ringold, G. M. (1989). Cationic Liposome-Mediated Transfection. Nature 337, 387-388.

Goodwin, E. C., and Rottman, F. M. (1992). The 3´-Flanking Sequence of the Bovine Growth Hormone Gene Contains Novel Elements Required for Efficient and Accurate Polyadenylation. J. Biol. Chem. 267, 16330-16334.

Izumi, M., Miyazawa, H., Kamakura, T., Yamaguchi, I., Endo, T., and Hanaoka, F. (1991). Blasticidin S-Resistance Gene (bsr): A Novel Selectable Marker for Mammalian Cells. Exper. Cell Res. 197, 229-233.

Kimura, M., Takatsuki, A., and Yamaguchi, I. (1994). Blasticidin S Deaminase Gene from Aspergillus terreus (BSD): A New Drug Resistance Gene for Transfection of Mammalian Cells. Biochim. Biophys. ACTA 1219, 653-659.

Kozak, M. (1987). An Analysis of 5´-Noncoding Sequences from 699 Vertebrate Messenger RNAs. Nucleic Acids Res. 15, 8125-8148.

Kozak, M. (1991). An Analysis of Vertebrate mRNA Sequences: Intimations of Translational Control. J. Cell Biology 115, 887-903.

Kozak, M. (1990). Downstream Secondary Structure Facilitates Recognition of Initiator Codons by Eukaryotic Ribosomes. Proc. Natl. Acad. Sci. USA 87, 8301-8305.

Lindner, P., Bauer, K., Krebber, A., Nieba, L., Kremmer, E., Krebber, C., Honegger, A., Klinger, B., Mocikat, R., and Pluckthun, A. (1997). Specific Detection of His-tagged Proteins With Recombinant Anti-His Tag scFv-Phosphatase or scFv-Phage Fusions. BioTechniques 22, 140-149.

Miller, J. H. (1972). Experiments in Molecular Genetics (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory).

Nelson, J. A., Reynolds-Kohler, C., and Smith, B. A. (1987). Negative and Positive Regulation by a Short Segment in the 5´-Flanking Region of the Human Cytomegalovirus Major Immediate-Early Gene. Molec. Cell. Biol. 7, 4125-4129.

Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press).


20

References, Continued

Shigekawa, K., and Dower, W. J. (1988). Electroporation of Eukaryotes and Prokaryotes: A General Approach to the Introduction of Macromolecules into Cells. BioTechniques 6, 742-751.

Takeuchi, S., Hirayama, K., Ueda, K., Sakai, H., and Yonehara, H. (1958). Blasticidin S, A New Antibiotic. The Journal of Antibiotics, Series A 11, 1-5.

Wigler, M., Silverstein, S., Lee, L.-S., Pellicer, A., Cheng, Y.-C., and Axel, R. (1977). Transfer of Purified Herpes Virus Thymidine Kinase Gene to Cultured Mouse Cells. Cell 11, 223-232.

Yamaguchi, H., Yamamoto, C., and Tanaka, N. (1965). Inhibition of Protein Synthesis by Blasticidin S. I. Studies with Cell-free Systems from Bacterial and Mammalian Cells. J. Biochem (Tokyo) 57, 667-677.

©2009, 2010 Life Technologies Corporation. All rights reserved. For research use only. Not intended for any animal or human therapeutic or diagnostic use.

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Notes

User Manual

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Documents

pcDNA 6/myc-His A, B, and C - Thermo Fisher Scientific · PureLink™ HiPure Plasmid Miniprep Kit 100 preps K2100-03 PureLink ... testing. The sequence of pcDNA™6/myc-His A is available